This protocol describes how to perform agarose gel electrophoresis.


Category
Protocol
Record History
Added on May 11, 2010 at 4:11 PM by Lindner, Jill
Modified on July 1, 2010 at 2:03 PM by Lindner, Jill
Shared with (contributions)
VCSCBi: Training / Protocols Master

Reagents, supplies, and equipment:

50X TAE (Tris-Acetic Acid-EDTA)

  • 242 g Tris base
  • 57.1 ml glacial acetic acid
  • 100 ml 0.5 M EDTA (pH 8.0)
  • q.s. to 1 L with DI H2O

0.5 M EDTA

  • 93 g disodium EDTA (Na2[EDTA])
  • 300 ml MilliQ H2O
  • adjust to pH 8.0 with 10 N NaOH (EDTA will not go into solution until pH < or = 7.0.)
  • q.s. to 500 ml with MilliQ H2O

Loading dye

  • 0.125 g bromophenol blue
  • 12.5 g ficoll - type 400
  • q.s. to 50 ml with MilliQ H2O

1X TAE

  • 20 ml 50 X TAE
  • q.s to 1 L with MilliQ H2O

1% agarose gel

  • 0.5 g agarose (Seakem LE Agarose #50004)
  • 50 ml 1X TAE
  • 2.5 ml ethidium bromide (this is a carcinogen)

Procedure:

  1. Place 50 ml 1X TAE in a 250 ml Erlenmeyer flask
  2. Add 0.5 g agarose. Do not mix.
  3. Microwave on medium heat for ~2 min.
  4. Remove from microwave and swirl.  If agarose is not dissolved, microwave again for ~ 30 sec.
  5. Cool on bench ~ 4 min or until you are able to touch the flask without discomfort from heat.
  6. Add 2.5 ml ethidium bromide and mix by gently swirling.
  7. Pour the agarose mixture into a 50 ml casting tray and insert the desired comb.
  8. Remove air bubbles with a pipette tip if necessary.
  9. Allow gel to set for ~30 min - 1 hr.
  10. Prepare a DNA standard that includes band sizes close to your expected band sizes by diluting 1 µl of standard in 9 µl running buffer plus 2 µl loading dye.
  11. Prepare sample by mixing 10 µl sample + 2 µl loading dye.  This may be performed in a 0.5 ml tube or on a piece of unused parafilm.  (Each well from a 15 tooth comb will hold ~ 12 µl.)
  12. Remove the gel from the casting tray and remove the comb from the gel.
  13. Pour ~ 250 - 300 ml (enough to cover the gel) running buffer into the gel electrophoresis box and gently place the gel into the box.
  14. Mix the standard plus dye by pipetting up and down several times and then load it into a well.  Mix the sample plus loading dye in the same manner and load the samples into individual wells.  The sample will displace the buffer in the wells.  Take care to keep the standard/sample in the wells.
  15. Once all the samples are loaded, place the lid on the gel box and connect the red and black leads from the power supply to the gel box.  DNA is positively charged and will run toward the positive (red) node.
  16. Turn on the power supply and run the gel at ~ 100 - 120V until the dye front is approximately halfway down the gel.  This will take ~ 25 - 30 min.
  17. Turn the power off, remove the lid and gel, and photograph the gel on a transilluminator.
  18. Once the gel is successfully photographed and/or the image is stored electronically, dispose of the gel in a hazardous wast container for ethidium bromide waste.